Enhanced stability of proteins immobilized on nanoparticles

ABSTRACT

This invention is directed to the application of a previously unknown property of nanomaterials—its ability to enhance protein activity and stability at high temperatures, in organic solvents, and in polymer composites. Nanomaterials such as single-walled carbon nanotubes (SWNTs) can significantly enhance enzyme function and stability in strongly denaturing environments. Experimental results and theoretical analysis reveal that the enhancement in stability is a result of the curvature of these nanoscale materials, which suppresses unfavorable protein-protein interactions. The enhanced stability is also exploited in the preparation of highly stable and active nanocomposite films that resist nonspecific protein absorption, i.e., inhibit fouling of the films. The protein-nanoparticles conjugates represent a new generation of highly selective, active, and stable catalytic materials. Furthermore, the ability to enhance protein function by interfacing them with nanomaterials has a profound impact on applications ranging from biosensing, diagnostics, vaccines, drug delivery, and biochips, to novel hybrid materials that integrate biotic and abiotic components.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/607,816, filed on Sep. 8, 2004. The entire teaching of the aboveapplication is incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant from theNational Science Foundation (DMR-0117792). The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Interfacing nanomaterials, in particular carbon nanotubes, withbiomolecules are important for applications ranging from biosensors,biorecognition probes, and molecular electronics to drug delivery. Amajor obstacle in the pursuit of applications of these conjugates stemsfrom the poor stability of biomolecules in harsh environments.

SUMMARY OF THE INVENTION

This invention is directed to protein compositions that comprisebiologically active proteins that are less susceptible to degradationthan normal. For example, this invention is directed to compositionsthat enzymatically act on substrates where the enzymes of thecompositions are less susceptible to degradation than normal. Theproteins and enzymes of these compositions can retain biological orenzymatic activity even when the compositions and substrates are innormally harsh or hostile environments, such as abnormal pHs,temperatures, high salinities, or media, including non-aqueous mediasuch as organic solvents, ionic liquids, gaseous media, andsupercritical fluids.

The compositions of this invention are proteins, e.g. enzymes, bound tothe external surfaces of nanoparticles. These nanoparticles haveexternal surfaces whose radius of curvature is commensurate with thedimensions of each of the proteins or enzymes, that are bound to thenanoparticles. When this size relationship is met, the stability of thebound proteins or enzymes is greater than the stability of theseproteins when they are bound to particles or surfaces whose radius ofcurvature is greater than the dimensions of each of the bound proteins,e.g., the proteins bound to flat surfaces. This stability differenceexists even when the material which forms the nanoparticles and the moreflat substrata are the same. The enhanced stability of the compositionsof this invention is maintained when the compositions are attached to amacroscopic surface or are embraced within the polymer.

This invention is also directed to methods of detecting analytes, evenwhen the analytes are in a solution that provides a harsh or hostileenvironment for enzymes. At least a portion of these analytes isnormally a substrate for the enzymes. The analyte detection methods ofthis invention utilize the compositions of this invention that containthe appropriate enzymes.

This invention is also directed to methods for preventing fouling ofsurfaces by fouling agents. These fouling agents are also substrates forenzymes and are often found in media that constitute a harsh or hostileenvironment for the enzymes. In this invention, the compositions of thisinvention are used to rid the media of these fouling agents byenzymatically degrading the agents, thereby keeping surfaces, which areoften fouled by the agents, free of these fouling agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1( a) is a bar graph showing the percent activity of soybeanperoxidase in its native state and on various supports in variousconcentrations of methanol.

FIG. 1( b) is a line graph showing the time-dependent deactivation ofsoybean peroxidase on various supports in 100% methanol.

FIG. 1( c) is a line graph showing the time-dependent deactivation ofsoybean peroxidase in its native state and on various supports in 95° C.aqueous solutions.

FIG. 1( d) is a line graph showing the time-dependent deactivation ofsubtilisin Carlsberg in its native state and on various supports atvarious temperatures.

FIG. 2( a) is a schematic showing soybean peroxidase on a flat support.

FIG. 2( b) is a schematic showing soybean peroxidase on a curvedsupport.

FIG. 2( c) is a line graph showing deactivation constants from soybeanperoxidase on various supports as a function of surface area coverage in95° C. aqueous solutions.

FIG. 2( d) is a line graph showing deactivation constants for soybeanperoxidase on various supports as a function of surface area coverage in100% methanol.

FIG. 2( e) is a bar graph showing deactivation constants for soybeanperoxidase on various supports at different amounts of surface areacoverage.

FIG. 2( f) is a micrograph of signal walled nanotubes on buckypaper.

FIG. 3( a) is a line graph showing deactivation constants for soybeanperoxidase on various supports in 95° C. aqueous solutions.

FIG. 3( b) is a line graph showing deactivation constants for soybeanperoxidase on various supports in 100% methanol.

FIG. 4( a) is a schematic showing the preparation of biocatalytic films.

FIG. 4( b) is a line graph showing the concentration-dependent activityof subtilisin Carlsberg on various supports on pMMA films.

FIG. 4( c) is a line graph showing the amount of human serum albuminadsorbed to pMMA films without and with subtilisin Carlsberg onsingle-walled nanotubes attached to the films.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

A core aspect of this invention is the formation of nanoparticles withproteins or enzymes attached to their external surfaces. Thesenanoparticles can be formed by a variety of techniques and from avariety of materials known in the art of nanoparticle fabrication. Thenanoparticles that are suitable in this invention generally includenanomaterials, e.g., nanotubes, nanosheets, nanoporous materials, suchas single-walled carbon nanotubes, multi-walled carbon nanotubes, goldnanoparticles or other metallic, semi-conducting, or metal oxidenanoparticles, quantum dots, functionalizes silica. Single-walled carbonnanotubes are preferred.

Proteins which can be used in this invention include proteins whichpossess a biological activity. A biological activity includescommercially relevant activities as a diagnostic, therapeutic, enzymaticor other protein activity. Examples of proteins includeimmunoglobulin-like proteins; antibodies; cytokines (e.g., lymphokines,monokines and chemokines); interleukins; interferons; erythropoietin;hormones (e.g., growth hormone and adrenocorticotropic hormone); growthfactors; nucleases; tumor necrosis factor; colony-stimulating factors;insulin; antigens (e.g., bacterial and viral antigens); DNA-bindingproteins and tumor suppressor proteins.

The enzymes of this invention can be of any type. The enzyme species isnot a critical aspect of the invention. Proteases, peroxidases, lipases,carbohydrate cleavage enzymes, carbohydrases, esterases, carboxylases,peroxidases, nucleases, lyases, ligases, isomerases, transferases, etc.can be used. The only requirements for each enzyme to be employed in theinvention are that it enzymatically acts on the substrate of interestthat is present in a solution to which the compositions of thisinvention are to be exposed, and that it be bindable to thenanoparticles of the compositions.

By way of example, transferases are enzymes transferring a group, forexample, the methyl group or a glycosyl group, from one compound(generally regarded as donor) to another compound (generally regarded asacceptor). For example, glycosyltransferases (EC 2.4) transfer glycosylresidues from a donor to an acceptor molecule. Some of theglycosyltransferases also catalyze hydrolysis, which can be regarded astransfer of a glycosyl group from the donor to water. The subclass isfurther subdivided into hexosyltransferases (EC 2.4.1),pentosyltransferases (EC 2.4.2) and those transferring other glycosylgroups (EC 2.4.99, Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (NC-IUBMB)).

Oxidoreductases catalyze oxido-reductions. The substrate that isoxidized is regarded as hydrogen or electron donor. Oxidoreductases areclassified as dehydrogenases, oxidases, mono- and dioxygenases.Dehydrogenases transfer hydrogen from a hydrogen donor to a hydrogenacceptor molecule. Oxidases react with molecular oxygen as hydrogenacceptor and produce oxidized products as well as either hydrogenperoxide or water. Monooxygenases transfer one oxygen atom frommolecular oxygen to the substrate and one is reduced to water. Incontrast, dioxygenases catalyze the insert of both oxygen atoms frommolecular oxygen into the substrate.

Lyases catalyze elimination reactions and thereby generate double bondsor, in the reverse direction, catalyze the additions at double bonds.Isomerases catalyze intramolecular rearrangements. Ligases catalyze theformation of chemical bonds at the expense of ATP consumption.

Hydrolases are enzymes that catalyze the hydrolysis of chemical bondslike C—O or C—N. The E.C. classification for these enzymes generallyclassifies them by the nature of the bond hydrolysed and by the natureof the substrate. Hydrolases such as lipases and proteases play animportant role in nature as well in technical applications ofbiocatalysts. Proteases hydrolyse a peptide bond within the context ofan oligo- or polypeptide. Depending on the catalytic mechanism proteasesare grouped into aspartic, serine, cysteine, metallo- and threonineproteases (Handbook of proteolytic enzymes. (1998) Eds: Barret, A;Rawling, N.; Woessner, J.; Academic Press, London).

Since the enzyme species is not a critical aspect of this invention, thesubstrate type is also not critical. Any substrate can be the target,provided it is enzymatically recognized by the enzyme species on thesurface of the nanoparticles of the compositions of this invention, andthat it is present in a solution to which the compositions of thisinvention are to be exposed.

The enzymes can be attached to the nanoparticles to form thecompositions of this invention by any suitable technique known in theart. Any chemical or physical bonding can be used. Hydrophobic bonding,hydrophilic bonding, ionic bonding, covalent and non-covalent bondingare suitable bonding types. Of these, hydrophobic bonding is preferred.A major consideration for the choice of bonding process to be employedis that the specific enzyme species bonded to the nanoparticles retain asubstantial fraction (e.g., at least about 30%, such as about 50%, atleast about 70% or more) of its native enzymatic activity after thebonding process has been completed.

A feature of the compositions of this invention can be that the surfacesof each of the nanoparticles to which the enzymes are attached have aradius of curvature that is within about 2, or preferably about 1,orders of magnitude of the dimensions of each attached enzyme. Thus, theradius of curvature of the nanoparticles is preferably about 100 nm orless.

The enzymes in the compositions of this invention are active when thecompositions are exposed to media, containing the substrates that areatypical for the enzymes in an isolated or unbound state. Although thecompositions of this invention exhibit very good enzymatic activity whenthe compositions are in physiological solutions containing the enzymesubstrate, they also exhibit very good activity and stability when themedia containing the substrate are considered to present harsh orhostile environments to the enzymes. For example, when the medium is anaqueous medium at an elevated temperature, e.g., greater than 90° C.,the enzymatic activity and stability of the compositions of thisinvention is maintained. If the medium is a hydrocarbon solvent, e.g.,an alcohol, the enzymatic stability of the compositions of thisinvention is greater than on more conventional (e.g., flat) surfaces.

The compositions of this invention have the advantage of retainingactivity when placed in liquid environments that are typically noxiousto the enzymes when these enzymes are not bound to the nanoparticlesthat are disclosed in this invention. For example, the compositions ofthis invention will be used in non-aqueous media, e.g., organicsolvents, ionic liquids, gaseous media and supercritical fluids, or inmedia at abnormal temperatures (e.g., other than 20° C. to 40° C.), ormedia where the pH is non-physiologically acidic or basic or in mediapossessing abnormal ionic strengths or salt levels (e.g., media withhigh salinity, such as sea water or a salt level of at least about 0.3 MNaCl). Of course, it is recognized that proteins that tolerate suchconditions are known (thermophilic enzymes, enzymes which tolerate highlevels of saline, etc). Thus, one can characterize harshness of anenvironment as a relative factor as compared to the normal reactionconditions of the enzyme. Thus, an abnormal temperature for athermophilic enzyme could be above the temperature at which thethermophilic enzyme is active. Likewise, an abnormal salinity for asalt-tolerant enzyme can be above the salinity levels at which thesalt-tolerant enzyme is active.

The compositions of this invention will also be used as antifoulingagents in paints, marine paints, coatings, lubricants, ointments, etc.These compositions are also intended for use as antimicrobial agents inenvironments where the antimicrobial activity of the bound enzymes isdesired for microbial lysis or inactivation.

Detection of analytes can be achieved by any number of procedures knownin the detection art. Formation of fluorescent species when enzymaticaction occurs, formation of absorption species when enzymatic actionoccurs, liberation of fluorescent or absorption tags when enzymaticaction occurs, formation of chemically reactive species by enzymaticaction that react with suitable target structures which thereby becomedetectable, formation of an electrically charged species by enzymaticaction which can be electrically detected are examples of detectionprocedures for analytes when the analytes are the targets of the boundenzymes in the compositions of this invention. Often, the analytes arelabels that have been attached to chemical moieties whose detection issought. In these instances, the analytes are the substrates for thebound enzymes in the compositions of this invention.

The compositions of this invention can be attached to macroscopicsurfaces or spread on or embedded within a polymeric material. Thecompositions of this invention can thereby be made functional parts ofuseful devices. The compositions of this invention can be added ascoatings to medical instruments, biosensors, biochips, biorecognitionprobes, biocatalytic films, biofuel cells, drug delivery systems,self-cleaning materials, resins, beads, and the like. These compositionscan be integral parts of permeable or nonpermeable membranes, sieves,tubing and the like. When incorporated in such devices, the compositionsof this invention can be used to detect analytes that are substrates ofthe enzymes bound to the nanoparticles, to monitor the presence ofsubstances in liquid media that are substrates of these enzymes, to actas antimicrobial agents when the enzyme substrates are integralconstituents of viral particles, microbial membranes or cell walls, orto prevent surface fouling by degrading substances that form deleteriousfilms on working surfaces of machines or instruments. There are manyutilities available to the skilled artisan for which the compositions ofthis invention are applicable. Enzymatic activity from solid materialsin a liquid environment is assumed to be one of the purposes for thecompositions of this invention. Improved enzyme stability when theliquid environment is normally hostile or harsh to the enzymes, whenthey are dissolved in the liquid, is of particular usefulness with thisinvention.

The emergence of techniques to generate nanomaterials with precisedimensions, geometries, and surface properties has resulted in anincreasingly large number of applications ranging from electronics andhigh-strength, lightweight materials to sensing elements. To date,proteins, and other biomolecules have been used to functionalizenanomaterials and influence their properties. However, up to now, verylittle is known about the ability of these nanoscale materials toenhance protein structure and function. Such information, however, is offundamental importance and is also critical for enhancing proteinfunction and stability on nanoparticles and therefore for designingoptimal protein-nanoparticle conjugates for use in functional materialsand surface coatings.

Materials and Methods

Enzymes and reagents. Soybean peroxidase, subtilisin Carlsberg, andN-succinyl-L-ala-L-ala-L-pro-L-phe-p-nitroanilide were purchased fromSigma as salt-free, dry powders and used without further purification.Raw single-walled nanotubes, SWNTs, were purchased from CarbonNanotechnologies, Inc., highly oriented pyrolytic graphite, HOPG SPI-2,was obtained from Structure Probe, Inc, and graphite was purchased fromAldrich. All the supports were used without further purification. Allother chemicals were purchased from Sigma and used as received.

Determination of enzyme activity. The initial rates of the phenolicoxidations catalyzed by SBP in presence of H₂O₂ were monitored byspectrophotometry. SC cleaves the peptide bond inN-succinyl-L-ala-L-ala-L-pro-L-phe-p-nitroanilide to release achromophore, p-nitroaniline, and the initial rates were obtained bymeasuring the increase in the absorbance at 405 nm.

Enzyme immobilization on SWNTs. The enzymes, Soybean peroxide (SBP) andsubtilisin Carisberg (SC) were adsorbed on SWNTs using hydrophobicinteractions. SWNTs were first sonicated in N, N-Dimethyl Formamide(DMF) for 20 minutes to obtain a uniform dispersion of SWNTs in DMF (1mg/mi). One ml of SWNT dispersion in DMF (i.e. 1 mg of SWNTs) was thendispensed in an Eppendorf micro-centrifuge tube and the organic phasewas gradually changed to an aqueous phase by repeated washing with pH 7buffer (50 mM phosphate). This gradual change from organic phase to anaqueous phase helps in a better dispersibility of SWNTs in buffer. Thedispersion of SWNT in pH 7 buffer was then exposed to freshly preparedsolutions of enzyme in buffer (pH 7 phosphate, 50 mM). This dispersionwas shaken on Innova™2000 (New Brunswick Scientific) platform shaker for2 h at 200 rpm at room temperature. In the case of SC, the shaking wascarried out at 4° C. to prevent autolysis of the protease duringincubation. After the 2 h incubation, the SWNTs were settled using amicro-centrifuge (Fisher Scientific) and the supernatant was removed.Typically, 6 washes were performed with fresh buffer to remove anyunbound/loosely bound enzyme. All supernatants were analyzed for proteincontent using the BCA or the μBCA assay (Pierce Biotechnology, Inc.). Itwas seen that the SWNTs interfere strongly with BCA/μBCA assay. Theamount of enzyme loaded on the SWNTs was, therefore, determined bymeasuring the concentration of enzyme solution before and after exposingit to the dispersion of SWNTs in buffer. The difference in the amount ofenzyme gives the amount of enzyme loaded on the SWNTs. A stable value ofenzyme loading on SWNTs was obtained by accounting for the loss ofenzyme due to leaching during the washes.

Determination of enzyme activity. The activity of SBP was measured usingthe p-Cresol assay. SBP catalyzes the oxidation of p-Cresol by H₂O₂ toform oligophenol and polyphenol products that fluoresce. For a typicalsolution phase assay, the reaction mixture consisted of 0.15 μg/mlsolution of SBP (made by serial dilution), 20 mM solution of p-Cresoland 0.125 mM solution of H₂O₂ all solutions were made in pH 7.0 buffer(phosphate, 50 mM). The initial rates of reaction were then measured bytracking the increase in fluorescence of the reaction mixture at anexcitation wavelength of 325 nm and emission wavelength of 402 nm usinga HTS 7000 Plus Bio Assay Reader (Perkin Elmer). For activitymeasurements in organic solvent phase, the solvents were added duringthe final wash to make solutions of 0.15 μg/ml solution of SBP in pH 7.0buffer (phosphate, 50 mM) containing the required amount of solvent inthe solution. The p-Cresol and H₂O₂ solutions were made in pH 7.0 buffer(phosphate, 50 mM) containing the required concentrations of solvent.

For measuring the activity of SBP immobilized on SWNTs (SWNT-SBP), awell-mixed dispersion of SWNT-SBP (1 mg/ml) was made in buffer and aknown amount of SWNT-SBP was dispensed by using serial dilution. For atypical experiment 0.5 μg to 1.5 μg of SWNT-SBP was used based on theloading of the SBP. The enzymatic activity was measured using 20 mMp-Cresol and 0.125 mM H₂O₂ in PH 7.0 buffer (phosphate, 50 mM). It wasobserved that some of the immobilized enzyme leached during the serialdilutions. To account for the effect of the enzyme that would leachduring the measurement of the activity of the immobilized enzyme, theSWNT-SBP suspension was washed 6 times more with the same dilutions andbuffer used in the final activity measurement. Since the amount ofleached enzyme during these washes was too low (<15 ng/ml) to bereliably detected by any of the protein measurement assays, the amountof protein was estimated by measuring the activity of the enzyme in thewashes. It was assumed that the activity of the leached enzyme was thesame as that of the solution phase enzyme. Using the value of specificactivity of the solution phase enzyme and the initial rate of reactionfor the enzyme in the wash solution, the amount of enzyme present in thewashes was calculated. The final loading of the enzyme on the SWNTs wascorrected for this amount of leached enzyme before calculating thespecific activity of the immobilized enzyme. After all the washes weredone, the SWNT-SBP were dispersed in pH 7.0 buffer (phosphate, 50 mM)and then exposed to the substrate solution so that the finalconcentrations of the substrates were 20 mM p-Cresol and 0.125 mM H₂O₂.The dispersion was shaken at 200 RPM at all times during the reactionusing Innova™2000 platform shaker to avoid problems due to diffusionlimitations. At fixed time intervals, the SWNTs were settled using amicro-centrifuge and the fluorescence of a 200 μl aliquot of thesupernatant was measured using the Bio Assay Reader. The aliquot wasthen replaced in the reaction mixture. A plot of H₂O₂ consumed versustime gives the initial rate of reaction and hence the activity of theSBP immobilized on the SWNTs. For activity measurements in solventphase, the p-cresol and H₂O₂ solutions were made in pH 7.0 buffer(phosphate, 50 mM) containing the required concentrations of solvent.After all the washes, the SWNT-SBP were dispersed in pH 7.0 buffer(phosphate, 50 mM) containing the required amount of solvent and thenexposed to the substrate solution. For 100% solvent phase, aqueousSWNT-SBP phase was gradually changed to the organic phase by repeatedwashing with 100% solvent. This treatment rendered the finalconcentration of water in the solvent to about 1-2%.

The activity of SC was measured usingN-succinyl-L-ala-L-ala-L-pro-L-phe-pnitoranilide (tetrapeptide)(Sigma-Aldrich) as the substrate. For a typical solution phase assay, 1μg/ml of freshly prepared SC solution pH 8.0 buffer (phosphate, 50 mM)was used with a 100 μM solution of tetrapeptide in pH 8.0 buffer(phosphate, 50 mM).

Subtilisin Carlsberg, which is a protease, cleaves the peptide bond inthe substrate to release the chromophore, p-Nitroaniline, which absorbsat 405 nm. The activity of the enzyme was measured by measuring theincrease in the absorbance of the reaction mixture at 405 nm using theBio Assay Reader. The activity of SC immobilized on SWNTs (SWNT-SC) wasmeasured using the same technique as that used for immobilized SBP (asdescribed above). For SWNT-SC, however, 4 μg to 50 μg of functionalizedSWNTs were used for the measurement of activity based on the loading ofthe SC. After performing 6 washes like those done for SWNT-SBP, theSWNT-SC were dispersed in pH 8 buffer (phosphate, 50 mM) and thenexposed to 100 μM tetrapeptide solution (final concentration). Thedispersion was kept well mixed by shaking at 200 RPM at all times duringthe reaction using the platform shaker. At fixed time intervals, theSWNTs were settled using a micro-centrifuge and the absorbance of thesupernatant was measured at 405 μm using the Bio Assay Reader. A plot ofconcentration of p-Nitroaniline versus time gives the initial rate ofreaction and hence the activity of the SC immobilized on the SWNTs. Theactivity measurements in the organic phase were performed as explainedabove for SBP.

Enzyme immobilization on other supports. The enzymes were also adsorbedon other supports including highly oriented pyrolytic graphite (HOPG),self-assembled monolayers (SAMs) of undecanethiolate on gold (Gold SAM),multi-walled carbon nanotubes (MWNTs), graphite powder (1-2 μm), SWNTfilms and MWNT films. HOPG SPI-2 samples were obtained from StructureProbe, Inc and fresh surfaces were exposed by peeling off the exposedlayers using a scotch tape. Self-assembled monolayers of undecanethiolwere assembled from 0.02 mM solutions in absolute ethanol for 12 h. Thesamples were then removed from the solution and rinsed thoroughly bysquirting with ethanol for several seconds. This rinsing was sufficientto remove any unbound thiols from the surface. The synthesis of SWNT andMWNT films was performed by first dispersing SWNTs and MWNTs in pH 7.0buffer (phosphate, 50 mM) as explained above and filtering the samplesthrough a 0.8 μm ATTP filter. The filter papers with the SWNT and MWNTcakes were dried and were attached to plastic troughs using appropriateclips. SBP and SC were adsorbed in HOPG, Gold SAM, SWNT films, MWNTfilms by dipping the supports into a solution of the enzymes and shakingthe samples on Innova™2000 (New Brunswick Scientific) platform shakerfor 2 h at 200 rpm at room temperature. In the case of SC, the shakingwas carried out at 4° C. to prevent autolysis of the protease duringincubation. The samples were then washed 6 times with pH 7.0 buffer(phosphate, 50 mM) to remove any loose/unbound enzyme. The loading onMWNTs was done as described above for SWNTs. After all the washes weredone the enzyme bound supports were exposed to the substrate solutionsand their activities were measured as outlined above for SWNTs.

The SWNT-enzyme conjugates were prepared in aqueous buffer by adsorbingtwo model enzymes subtilisin carlsberg (SC) and soybean peroxidase (SBP)onto SWNTs. The enzymes showed strong affinity for SWNTs with saturationlevels of 670 and 655 μg/mg SWNT for SC and SBP, respectively. Both SCand SBP retained a substantial fraction of their native enzymaticactivity; specific activities of the adsorbed SC and SBP in aqueousbuffer were ca. 63% and ca. 38% of the native enzyme activitiesrespectively. FT-IR spectroscopy analysis. revealed ca. 11.5% and ca.13% total change in the secondary structure of SC and SBP respectivelydue to absorption onto SWNTs. AFM studies also revealed that both SBPand SC retained their tertiary structure on adsorption on SWNTs. Thissuggests that the present method employed for interfacing SBP and SCwith SWNTs results in a minimal loss in the native structure.

Hammett analysis was used as a sensitive probe of transition statestructure and enzyme mechanism. The Hammett coefficient (ρ) provides ameasure of the sensitivity of SBP's catalytic efficiency to theelectronic nature of substituents on phenolic substrates. Hammettanalysis revealed ρ values of 1.7±0.21 and 1.4±0.12 for SWNT-SBP andHOPG-SBP, respectively, in 100% methanol. The comparable values of ρsuggest that the mechanism of catalysis is similar for SBP adsorbed onthe two supports; the significantly greater retention of activity forSBP immobilized on SWNTs than for SBP immobilized on HOPG in 100%methanol is therefore not due to a change in the mechanism of catalysison the different supports.

It was also found that a variety of proteins differing in both structureand function, including horseradish peroxidase, subtilisin carlsberg,proteinase K, trypsin, and lipase, remain catalytically active uponadsorption onto SWNTs, with specific activities ranging from 40-70%relative to that of the native protein in aqueous buffer.

Having established that a number of enzymes retain activity on SWNTs inaqueous buffer, SWNTs were examined to determine whether the enzymesfunction in strongly denaturing environments—environments in whichnative enzymes show poor retention of activity. To that end, SWNT-SBPwas added to solutions of buffer containing the denaturant methanol.FIG. 1 a shows the retention of activity in solutions containingmethanol, i.e. the enzymatic activity in solutions containing methanolnormalized to the enzymatic activity in aqueous buffer, for native SBPand SBP adsorbed onto a variety of supports. The specific activity—theactivity normalized to the amount of enzyme—for native SBP and thevarious SBP conjugates in solutions containing methanol was alsodetermined. Native SBP was completely inactive in 100% methanol.However, the SWNT-SBP conjugates retained relatively high catalyticactivity, even in neat methanol (FIG. 1 a). It is well known thatproteins are often stabilized by immobilization onto a support. Toassess whether the stabilization of SBP on SWNTs was simply a result ofimmobilization, the enzyme was absorbed onto graphite flakes. Since aSWNT is similar to a rolled graphene sheet, graphite flakes represent anideal surface for comparison. As shown in FIG. 1 a, SBP wassignificantly more active on SWNTs than on graphite flakes, particularlyin neat methanol. A similar trend was also observed in isopropanol,trifluoroethanol, and acetonitrile (data not shown).

The SWNT-SBP conjugates were also more active in methanol than enzymeimmobilized onto a variety of other flat supports, including highlyordered pyrolytic graphite (HOPG) and self-assembled monolayers (SAMs)of undecanethiolate on gold-coated glass cover slips (FIG. 1 a).Finally, SWNT films were prepared by filtering a suspension of SWNTsthrough a 0.8 μm membrane. SBP adsorbed onto the resulting “SWNTbuckypaper” was more active than SBP adsorbed on the flat supports (FIG.1 a), suggesting significantly different behavior under denaturingconditions on these nanoscale supports relative to flat surfaces.Similar results were obtained for multi-walled carbon nanotubes, andgold particles of similar dimensions as the SWNT (data not shown).

In addition to the initial activity, the stability of the SWNT-enzymeconjugates was evaluated in strongly denaturing environments. Thehalf-life of SBP adsorbed onto SWNTs in 100% methanol was at leasttwo-fold longer than that of the enzyme adsorbed onto flat supports(FIG. 1 b). The thermostability of the conjugates was also tested at 95°C., a temperature at which native SBP undergoes significant and rapiddenaturation. The half-life of SBP adsorbed onto SWNTs at 95° C. wasapproximately 90 min, ten-fold longer than that of the native enzyme andat least twice that of SBP adsorbed onto other supports (FIG. 1 c). Asimilar enhancement in stability was seen for SBP adsorbed onto SWNTbuckypaper (data not shown). These results indicate a dramaticenhancement in stability in harsh environments for SBP adsorbed ontoSWNTs. The observed stabilization on SWNTs is not unique to SBP, but isalso seen for the unrelated protease subtilisin Carlsberg (SC) (FIG. 1d).

To see if SWNTs render SC more resistant to degradation by autolysis,the storage stability of SWNT-SC and native SC was examined at twodifferent conditions—pH 7.8, at which the protease is most active and pH4.5, at which the proteolytic activity of SC is negligible. FIG. 3 bshows that at both pH conditions, the loss in activity of SC adsorbed onSWNTs is less than that of SC adsorbed on HOPG. The half life of HOPGSCin pH 7.8 was ca. 44 h, about two fold lower than that of SWNT-SC.Interestingly, the activities of adsorbed SC are similar for both the pHconditions, which shows that the loss in activity over time is not dueto autolysis, but probably due to protein-surface or protein-proteininteractions on the surface of the hydrophobic supports. This furtherdemonstrates the impact of the nanoscale environment on the reportedenhanced stability of SWNT-enzyme conjugates.

There are three possible hypotheses that could explain the enhancedstability of enzymes on SWNTs. The first (hypothesis 1) is that proteindeactivation in harsh environments is primarily mediated byprotein-surface interactions, which are disfavored on highly curvedsupports such as SWNTs relative to flat supports. An alternativehypothesis (hypothesis 2) stems from the observation that the greaterstability of adsorbed enzymes relative to their soluble counterparts isdue to greater barriers to unfolding on the supports, as a result ofprotein-support interactions. Therefore, if proteins have a higheraffinity for SWNTs than for other supports, there may be greaterbarriers to unfolding in harsh environments on SWNTs than on othersupports, thereby explaining the higher stability observed on SWNTs.Finally, a third hypothesis is that lateral interactions betweenadsorbed proteins contribute to protein deactivation in harshenvironments, and that these unfavorable “lateral” interactions aresuppressed on highly curved supports such as SWNTs relative to those onflat surfaces (FIGS. 2 a and b). This third hypothesis is explained ingreater detail below.

FIG. 2 a depicts proteins adsorbed on a flat support, where x and yrepresent the distances between adjacent proteins (measured along theprotein-substrate interface) along the X and Y axes, respectively.Similarly, x_(f)′ and y_(f)′ represent the center-to-center distancebetween adjacent proteins along the X and Y-axes, respectively. On aflat support x=x_(f)′, and y=y_(f)′. Furthermore, the surface coverageof proteins is inversely proportional to the product xy. FIG. 2 bdepicts proteins adsorbed on a cylindrical support, where x and yrepresent the distances between adjacent proteins (measured along theprotein-substrate interface) along the circumference (θ-direction) andthe axis of the cylinder, respectively. Here, the values of x and y areidentical to those in FIG. 2 a. Finally, x_(c)′ and y_(c)′ represent thecenter-to-center distance between adjacent proteins along thecircumference and the axis of the cylinder, respectively. On acylindrical support, y_(c)′=y; however, x_(c)′ is not equal to x, but isgreater than x.

A simple geometric analysis (equation 1) reveals that,

$\begin{matrix}{x_{c}^{\prime} = {\frac{\left( {R + r} \right)}{R}*x}} & (1)\end{matrix}$

where R is the radius of the cylinder, and r represents the averagedimension of SBP. Consequently, at the same separation along theprotein-substrate interface, and the same surface coverage, thecurvature of a cylindrical support results in an increase in thecenter-to-center distance between adjacent proteins (FIG. 2 b). Ifunfavorable interactions between adjacent proteins contribute to theirdeactivation in harsh environments, then this increase in separationshould result in a decrease in the rate of deactivation, and couldcontribute to the greater protein stability on SWNTs relative to flatsupports.

Both experimental data (FIGS. 2 c-e) and theoretical analysis (seediscussion below and FIGS. 3 a-b) are used to distinguish among thesehypotheses. The rates of deactivation were measured in aqueous buffer at95° C. and in methanol for SBP adsorbed onto SWNTs and graphite flakesat different fractional surface coverages. FIGS. 2 c and d reveal astrong dependence of the enzymatic deactivation rate on surfacecoverage, with identical deactivation constants on SWNTs and graphiteflakes should persist even at low coverages. Similarly, if the enhancedstability on SWNTs is a result of a greater affinity of the protein forSWNTs (hypothesis 2), the difference in stability should also persist atlow surface coverages. The results shown in FIGS. 2 c and d are clearlyinconsistent with hypotheses 1 and 2, yet they are consistent withhypothesis 3. If unfavorable “lateral” interactions between adsorbedproteins contribute significantly to protein deactivation (hypothesis3), these interactions, and hence the rate of enzymatic deactivation,should decrease on all supports with decreasing surface coverage (i.e.with an increase in the average separation between adsorbed proteins).Furthermore, hypothesis 3 also predicts that the enhancement instability on SWNTs relative to graphite flakes should disappear at verylow surface coverages.

Additional control experiments were performed to confirm that thesimilar values of the deactivation constants on SWNT and graphite flakesat low surface coverage are a result of a reduction in unfavorablelateral interactions, and not due to a change in the conformation of theadsorbed protein at low surface coverage. For this purpose, the rates ofenzymatic deactivation at 95° C. (FIG. 2 e) were measured for thefollowing sets of protein conjugates: 1) SBP adsorbed onto SWNTs andgraphite flakes at a high fractional surface coverage (0.75); 2) SBPadsorbed onto SWNTs and graphite flakes at a low fractional surfacecoverage (0.07); and 3) SBP adsorbed onto SWNTs and graphite flakes at alow fractional surface coverage of 0.07 (same as that for sample set 2),followed by the adsorption of catalytically inactive apo-SBP, yielding afinal fractional surface coverage of 0.75 (same as that for sample set1). While preparing sample set 3, the active protein was allowed toadsorb prior to adsorbing the inactive apo-protein, thereby allowing itto change its conformation on the support (under conditions of lowcoverage). Furthermore, sample sets 2 and 3, contain the same amount of“active” protein, but differ in the total surface coverage of protein.

As seen in FIG. 2 e, the deactivation rate for SWNT-SBP conjugate 3 at95° C. is identical to that for SWNT-SBP conjugate 1, and is higher thanthat for SWNT-SBP conjugate 2. Similar trends are seen for thegraphite-SBP conjugates. Furthermore, the deactivation rate for theSWNT-SBP conjugate 3 is significantly lower than that for thegraphite-SBP conjugate 3. In combination, these results suggest that thedecrease in deactivation rate at low surface coverage (FIGS. 2 c and d)is not due to a change in the conformation of the adsorbed protein, andprovide further support for hypothesis 3.

Finally, a simple model has been developed that allows thequantification of the effect of substrate curvature on the lateralinteractions between adsorbed proteins. The model assumes that theproteins are distributed uniformly on the surface. A new variable, S,was introduced to capture the average center-to-center distance betweenadjacent proteins on the various supports. The term S is defined to bethe geometric mean of the center-to-center distances between proteinsalong the two orthogonal axes; i.e. S=(x_(f)′.y_(f)′)^(1/2) on a flatsupport, and S=(x_(c)′.y_(c)′)^(1/2) on a cylindrical support (FIGS. 2 aand b). As discussed above, for the same values of x and y, the value ofx_(c)′ is greater than the value of x_(f)′, and therefore the value of Son a cylindrical support is greater than that on a flat support. S maybe converted to a dimensionless form (ε) by dividing it by the value ofS on a flat support at maximum surface coverage (S_(m)); ε=S/S_(m).FIGS. 2 a and b indicate that S_(m) is equivalent to the geometric meanof the distance between adjacent proteins along the protein-substrateinterface along the two orthogonal axes, measured at maximum surfacecoverage.

The deactivation rates of adsorbed SBP on the various supports,previously plotted as a function of surface coverage (FIGS. 2 c and d),are now plotted as a function of the dimensionless variable ε (FIGS. 3 aand b). The introduction of ε, which now accounts for the curvature ofthe support, allows the data for the deactivation rates on graphiteflakes and SWNTs to collapse onto a single curve, providing furtherevidence in support of the “lateral-interaction” hypothesis.

The aforementioned model also predicts that the observed enhancement instabilization should not be unique to SWNTs. Consistent with thisprediction, an enhancement in the stability of proteins in harshenvironments on other nanostructured supports, including goldnanoparticles, was observed in the experiments described above. Thisphenomenon results from the radius of curvature of the nanoscale supportbeing commensurate with the dimensions of the protein, as illustratedschematically in FIG. 2 b. Consistent with this hypothesis, a scanningelectron micrograph of SWNT buckypaper (FIG. 2 f) shows SWNT bundleshaving an average diameter of ca. 8 nm. Moreover, the value of SBP'ssaturation loading on SWNTs (measured to be 655 mg/g) supports thisvalue of the average bundle diameter (16, 23). The average bundle radiusis therefore similar to the dimensions of SBP (6.1 nm×3.5 nm×4.0 nm).

These highly stable and active enzyme-nanotube conjugates are ideallysuited for designing functional nanocomposites; composites incorporatingenzymes, particularly proteases, may be useful for designinganti-fouling or self-cleaning surfaces. Previous applications have beenlimited by enzyme leaching from the matrix, low enzyme loading, and lowactivity of the incorporated enzymes because of poor stability in theharsh abiotic environment. The stable SWNT-enzyme composites should formhighly stable biocatalytic films. To that end, SWNT-SC conjugates weredispersed in poly(methyl methacrylate) (pMMA) (FIG. 4 a) and theenzymatic activities of the films were measured. The pMMA-SWNT-SC filmsretained >90% of their initial activity over 30 days in aqueous buffer.Furthermore, the high surface area per unit weight of the SWNTs resultedin high enzyme loadings in the films, and consequently, the films wereover 30 times more active than those containing identical amounts ofgraphite-SC conjugates (FIG. 4 b). The control pMMA-SC films exhibitedsignificant leaching, resulting in a nearly complete loss of activityafter washing (FIG. 4 b).

The proteolytic activity of the biocatalytic films will allow thepreparation of antifouling and antimicrobial surfaces, for example thosethat may be used on surgical instruments, implants, diagnostics,bioreactors, and other surfaces prone to contamination. The attachmentof bacteria to surfaces, which can result in the buildup of biofilms, isoften mediated by protein adsorption that will most likely be preventedby making surfaces protein-resistant. Biocatalytic nanocomposites thatresist non-specific protein adsorption were designed (FIG. 4 c). Toassess the protein resistance of these materials, the biocatalytic filmswere exposed to concentrated solutions (1 mg/mL) of the plasma proteinhuman serum albumin (HSA) continuously for 6 days. pMMA-SWNT-SC filmswere able to reduce the nonspecific binding of HSA by ca. 75% whencompared to films without SC (FIG. 4 c). SDS-PAGE of HSA incubated withpMMA-SWNT-SC films revealed proteolytic breakdown products (data notshown), suggesting that this decrease in HSA binding is due to theproteolytic degradation of the bound HSA and the subsequent desorptionof the peptide fragments rendering the film self-cleaning. Biocatalyticfilms that also incorporated a second protease, trypsin (TRY), adsorbedonto SWNTs to provide a broader range of proteolytic cleavage sites onthe HSA, yielded further reduction in the extent of nonspecific proteinadsorption, with as much as ca. 95% lower binding when compared with theenzyme-free film (FIG. 4 c). The biocatalytic films demonstrate the highstability required for continuous operation in commercial environments.When challenged with a fresh sample of HSA (1 mg/mL) every 3 days, thepMMA-SWNT-enzyme conjugates led to ˜50 fold decrease in HSA binding over30 days. These films are also active in high salt buffers and atelevated temperatures.

A thermal analysis was used to examine the pMMA films to ensure that thechanges in the physical properties of the polymer are minimal due to theincorporation of the SWNT-enzyme conjugates. The films were prepared asbefore and the glass-transition temperature (Tg) of the films weremeasured. The Tg values of pMMA and pMMA-SWNT-SC films were similar; xand y respectively.

In addition to polymeric composites, films composed solely of SWNTs andenzymes were prepared by filtering suspensions of SWNT-SC and SWNT-TRYconjugates through a 0.8 μm membrane (FIG. 4 a). These “biocatalyticbuckypapers” have enzyme loadings as high as 30% (w/w), which are amongthe highest loadings reported to date. As shown in FIG. 4 c, these filmsshowed negligible protein adsorption after 6 days (>99% reduction in theamount of protein adsorption compared to the enzyme-free film).

The enhanced stability of proteins adsorbed on nanotubes, in addition tothe other attractive features (minimal leaching, high surface area perunit weight, and high strength to name a few) will thus be used inapplications ranging from biosensing to biomedical devices, which wouldrequire highly stable protein-nanotube conjugates. The experimentalresults and the accompanying theoretical analysis shown here indicatethat the observed enhancements in protein stability are not unique tonanotubes and will also be obtained with other nanomaterials. Theability to enhance protein function by interfacing them withnanomaterials will have profound impact on the design of biosensors,biorecognition probes, protein chips, biofuel cells, vaccines, novelcomposites and supports for biotransformations, drug delivery systems,and self-cleaning materials.

Figure Legends

FIG. 1 Retention of enzymatic activity when exposed to harshenvironments. (a) The initial activity (v) in solutions containingmethanol relative to the activity in aqueous buffer for native SBP (1)and SBP adsorbed on various supports—SWNTs (2), buckypaper made out ofSWNTs (3), HOPG (4), and graphite flakes (5). The asterisk indicates noactivity of the native SBP in 100% (<0.01% H₂O) methanol. (b)Time-dependent deactivation of SBP in 100% methanol on varioussupports—SWNTs (1), HOPG (2), SAM of undecanethiolate on gold (3), andgraphite flakes (4). For (b)-(d), the activities are normalized relativeto the initial activity (activity at t=0 min), and each data pointrepresents an average of triplicate measurements with standard error<10%. (c) Time-dependent deactivation of SBP at 95° C. on varioussupports—native SBP (1 open circles), SWNTs (2 black circles), HOPG (3diamonds), SAM of undecanethiolate on gold (4 open squares), andgraphite flakes (5 triangles). (d) Time-dependent deactivation of SC onvarious supports in aqueous buffer—native SC (open circles), SC adsorbedon SWNTs (black circles) at 50° C. (open circles) and 70° C. (opentriangles).

FIG. 2 Effect of lateral interactions on the deactivation constants ofSBP adsorbed onto different supports. (a) Schematic depicting SBPmolecules adsorbed onto a “flat” support. (b) Schematic (drawnapproximately to scale) depicting SBP molecules adsorbed onto acylindrical support. The curvature of the support increases thecenter-to-center distance between adjacent proteins (x_(c)′), relativeto the distance on a “flat” support (x_(f)′). (c) Deactivation constantsfor SBP adsorbed onto SWNTs (circles) and graphite flakes (triangles) asa function of surface coverage at 95° C. In (c) and (a), error barsindicate the standard deviation of triplicate measurements. (a)Deactivation constants for SBP adsorbed onto SWNTs (circles) andgraphite flakes (triangles) as a function of surface coverage in 100%methanol. (e) Deactivation constants for SBP adsorbed onto SWNTs andgraphite flakes at a fractional surface coverage of 0.75 (black bars), afractional surface coverage of 0.07 (hatched bars), and a fractionalsurface coverage of 0.07 for SBP, with a “total” surface coverage of0.75 for SBP and apo-SBP (gray bars). (f) SEM image of a typical SWNTbuckypaper.

FIG. 3 Influence of the center-to-center distance between adsorbedproteins on the deactivation rate. Deactivation constants for SBPadsorbed onto SWNTs (circles) and graphite flakes (triangles) as afunction of the dimensionless variable ε (a) at 95° C. and (b) in 100%methanol. Error bars indicate the standard deviation of triplicatemeasurements. The introduction of the dimensionless variable ε enablesthe deactivation rates on different supports to be collapsed onto asingle curve.

FIG. 4

Enzymatic activities of biocatalytic films. (a) Preparation ofbiocatalytic films along with a SEM image of a typical biocatalyticbuckypaper. (b) Activities of native SC (1), SC adsorbed on SWNTs (2),and SC adsorbed on graphite powder (3) in pMMA films as a function ofthe amount of SC conjugates loaded into the films. The activities weremeasured after the films were washed extensively with aqueous buffer.(c) Protein-resistant properties of the biocatalytic films—amount of HSAadsorbed onto plain pMMA films (control, 1), pMMA-SWNT-SC films (2),pMMA-SWNT-SC-TRY films (3) and SWNT-SC-TRY buckypaper (4). Error barsindicate the standard deviation of triplicate measurements.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A composition comprising: (a) nanoparticles; and (b) proteins,wherein said proteins are bound to said nanoparticles and saidnanoparticles have external surfaces whose radius of curvature is within2 orders of magnitude of the dimensions of each of said proteins boundto said nanoparticles, such that the stability of said bound proteins isgreater than the stability of said proteins bound to surfaces of thesame material as that of said nanoparticles but which forms a flatsurface.
 2. The composition of claim 1, wherein said protein stabilityis higher than on a flat support in a liquid medium other than anaqueous medium at neutral pH, an aqueous medium at normal salinity, oran aqueous medium at a temperature between about 20° C. and 40° C. 3.The composition of claim 2, wherein said liquid medium is selected fromthe group consisting of an aqueous medium at a temperature greater thanabout 40° C., less than about 10° C., an aqueous medium whose pH is lessthan about pH 6.5, an aqueous medium whose pH is greater than about pH7.5, an aqueous medium with a salinity of at least about 0.3 M NaCl, anon-aqueous medium, and combinations thereof.
 4. The composition ofclaim 1, wherein said nanoparticles are selected from the groupconsisting of single-walled carbon nanotubes, multi-walled carbonnanotubes, gold or other metallic nanoparticles, semi-conductingnanoparticles, metal oxide nanoparticles, quantum dots, funtionalizedsilica, and mixtures thereof.
 5. The composition of claim 1, whereinsaid proteins are bound to said nanoparticles through hydrophobicbonding, hydrophilic bonding, ionic bonding, covalent bonding, andnon-covalent bonding.
 6. The composition of claim 1, wherein saidprotein is an enzyme.
 7. An article of manufacture comprising thecomposition of claim 1 bound to a macroscopic surface.
 8. The article ofmanufacture of claim 7, wherein said macroscopic surface is selectedfrom the group consisting of a polymer, a polymeric film, a metal, ametal alloy, and combinations thereof.
 9. The article of manufacture ofclaim 7, wherein said article is incorporated in a member of the groupconsisting of a biosensor, a biochip, a biofuel cell, a drug deliverysystem, an antimicrobial film, a paint antifouling film, and a lubricantantifouling film.
 10. A method of making a device containing acomposition which can enzymatically act on one or more substances in asolution comprising: (a) bonding one or more enzyme species tonanoparticles, wherein said nanoparticles have external surfaces whoseradius of curvature is within 2 orders of magnitude of the dimensions ofeach said enzyme bound to each said nanoparticle, such that the activityof said enzymes is greater than the activity of said enzymes bound tosurfaces of the same material as that of said nanoparticles but whichforms a flat surface, thereby forming said composition; and (b)attaching said composition to a working surface of said device wheresaid working surface will be in contact with said solution when theenzyme activity of said enzymes is desired; thereby forming the device.11. The method of claim 10, wherein the solvent of said solution is notan aqueous medium at neutral pH, an aqueous medium at normal salinity,or an aqueous medium at a temperature between about 20° C. and 40° C.when said working surface is in contact with said solution.
 12. Themethod of claim 11, wherein said solvent is selected from the groupconsisting of an aqueous medium at a temperature greater than about 40°C., less than about 10° C., an aqueous medium whose pH is less thanabout pH 6.5, an aqueous medium whose pH is greater than about pH 7.5, aliquid hydrocarbon medium, an aqueous medium with a salinity of at leastabout 0.3 M NaCl, and combinations thereof.
 13. The method of claim 10,wherein said nanoparticles are selected from the group consisting ofsingle-walled carbon nanotubes, multi-walled carbon nanotubes, gold orother metallic nanoparticles, semi-conducting nanoparticles, metal oxidenanoparticles, quantum dots, funtionalized silica, and mixtures thereof.14. The method of claim 10, wherein said enzymes are bound to saidnanoparticles through hydrophobic bonding, hydrophilic bonding, ionicbonding, covalent bonding, and non-covalent bonding.
 15. A method ofdetecting an analyte in a solution comprising: (a) contacting saidsolution containing said analyte with a composition comprising (i)nanoparticles, and (ii) enzymes, wherein said enzymes are bound to saidnanoparticles and said nanoparticles have external surfaces whose radiusof curvature is within 2 orders of magnitude with the dimensions of eachsaid enzyme bound to said nanoparticles, such that the activity of saidbound enzymes is greater than the activity of said enzymes bound tosurfaces of the same material as that of said nanoparticles but whichforms a flat surface, and further wherein said analyte is a substratefor said enzymes; (b) allowing said enzymes to enzymatically act on saidanalyte, thereby forming a product that is detectable by external means;and (c) detecting said product by said external means, thereby detectingsaid analyte.
 16. The method of claim 15, wherein said enzyme activityis maintained in a liquid medium other than an aqueous medium at neutralpH, an aqueous medium at normal salinity, or an aqueous medium at atemperature between about 20° C. and 40° C. when said working surface isin contact with said solution.
 17. The method of claim 16, wherein saidliquid medium is selected from the group consisting of an aqueous mediumat a temperature greater than about 40° C., less than about 10° C., anaqueous medium whose pH is less than about pH 6.5, an aqueous mediumwhose pH is greater than about pH 7.5, a liquid hydrocarbon medium, anaqueous medium with a salinity of at least about 0.3 M NaCl, andcombinations thereof.
 18. The method of claim 15, wherein saidnanoparticles are selected from the group consisting of single-walledcarbon nanotubes, multi-walled carbon nanotubes, gold or other metallicnanoparticles, semi-conducting nanoparticles, metal oxide nanoparticles,quantum dots, funtionalized silica, and mixtures thereof.
 19. The methodof claim 15, wherein said enzymes are bound to said nanoparticlesthrough hydrophobic bonding, hydrophilic bonding, ionic bonding,covalent bonding, and non-covalent bonding.
 20. A method of reducing thefouling of a surface by a substance present in a solution comprising:(a) contacting said solution containing said substance with said surfacewherein a composition is attached to said surface, said compositioncomprising (i) nanoparticles, and (ii) enzymes, wherein said enzymes arebound to said nanoparticles and said nanoparticles have externalsurfaces whose radius of curvature is within 2 orders of magnitude ofthe dimensions of each said enzyme bound to said nanoparticles, suchthat the stability of said bound enzymes is greater than the stabilityof said enzymes bound to surfaces of the same material as that of saidnanoparticles but which forms a flat surface, and further wherein saidsubstance is a substrate for said enzymes; and (b) allowing said enzymesto enzymatically degrade said substance, thereby reducing the amount ofsaid substance in said solution and the fouling adherence of saidsubstance to said surface.
 21. The method of claim 20, wherein saidenzyme activity is maintained in a liquid medium other than an aqueousmedium at neutral pH, an aqueous medium at normal salinity, or anaqueous medium at a temperature between about 20° C. and 40° C. whensaid working surface is in contact with said solution.
 22. The method ofclaim 21, wherein said liquid medium is selected from the groupconsisting of an aqueous medium at a temperature greater than about 40°C., less than about 10° C., an aqueous medium whose pH is less thanabout pH 6.5, an aqueous medium whose pH is greater than about pH 7.5, aliquid hydrocarbon medium, an aqueous medium with a salinity of at leastabout 0.3 M NaCl, and combinations thereof.
 23. The method of claim 20,wherein said nanoparticles are selected from the group consisting ofsingle-walled carbon nanotubes, multi-walled carbon nanotubes, gold orother metallic nanoparticles, semi-conducting nanoparticles, metal oxidenanoparticles, quantum dots, funtionalized silica, and mixtures thereof.24. The method of claim 20, wherein said enzymes are bound to saidnanoparticles through hydrophobic bonding, hydrophilic bonding, ionicbonding, covalent bonding, and non-covalent bonding.